1. Field of the Invention
The present invention relates to an apparatus for measuring the quantities of chemical substances contained in a solution to be assayed by using a chemical-substance sensitive sensor consisting of a potentiometric electrode which responds selectively to chemical substances, and a liquid-junction type reference electrode.
2. Description of Prior Art
Recently, in the physiological and medical fields, measurements are frequently made of the concentration of substances contained in the body fluids represented by the blood, for example ions such as hydronium ion, sodium ion, potassium ion, calcium ion, and chloride ion; gases such as oxygen and carbon dioxide; sugar such as glucose and lactose; and chemical substances such as hormones, enzymes, and antibodies. Various types of potentiometric electrodes have been used in the past. For the measurement of such chemical substances such as devices employing a glass electrode, a coated-wire electrode, and chemically modified metal electrode. Glass electrodes, in particular, are widely used, since they can be adapted to respond selectively to various kinds of chemical substances by changing the composition of the glass membrane or coating a chemically sensitive film on the glass membrane. However, the miniaturization of a glass electrode for the determination of the local amount of chemical substances in a tissue of a living body involves problems in that reduced strength of the glass membrane results and also in increased resistance of the glass membrane, which naturally leads to a decrease in the response speed.
As a miniature ion sensor capable of solving the aforesaid problem with such glass electrode, an ion sensor utilizing the field effect of semiconductors has been proposed in U.S. Pat. No. 4,020,830 and U.S. Pat. No. 4,218,298. This ion sensor is called ISFET (Ion Sensitive Field Effect Transistor). The boundary electric potential appearing on the surface of the ion sensitive membrane of said ISFET is dependent upon the activity of the specific ions contained in the solution to be assayed. A change in the boundary electric potential leads to a corresponding change in the conductivity of the channel under the ion sensitive membrane, if the electric potential of the solution is kept constant by means of a reference electrode. Where such principle is applied in detecting a boundary electric potential, the problem of electrode resistance as experienced with the conventional glass electrode is solved the output impedance involved is low by virtue of the impedance converting function of the ISFET; therefore, a high-input impedance amplifier as an external circuit is not required. This new-type ion sensor has the following characteristics:
(1) Since it involves little problem, if any, of electrode resistance, the ion sensor can be miniaturized so as for it to be inserted into a living body and is able to respond fast.
The IC technique used permits integration (multiplexing) of various types of ion sensors on one silicon chip.
(3) It is suitable for mass production.
Because of these characteristics the ISFET, as a sensor for monitoring the quantity of chemical substances in a living body by inserting it into the tissue of the body, is attracting wide attention. Further, it is known that the ISFET can be adapted to respond to various kinds of substances by modifying its ion-sensitive membrane. For example, there are the following types of ISFET: a pH sensor using silicon nitride, alumina, or tantalum pentoxide for the sensitive membrane; a cation (Na.sup.+, K.sup.+ or the like) sensor using an inorganic glass membrane; a chloride ion sensor having a sensitive membrane of an inorganic compound such as AgCl; an ion (Na.sup.+, K.sup.+, Ca.sup.++ or the like) sensor having a sensitive membrane formed of a polymer matrix, such as polyvinyl chloride or silicone resin, and crown ether, phosphate, or the like added or fixed thereto; and such other sensors for measuring substrates and immune substances as enzyme sensor and immuno-sensor, which comprise a combination of any of above named sensors and an enzyme membrane, an antibody or antigen.
An electric circuit as shown in FIG. 1 is usually employed in measuring the concentration of ions contained in the solution to be assayed by means of such ion sensor. An ISFET 1, together with a liquid-junction reference electrode 4, is immersed in the to-be-assayed solution 8 in a receptacle 6. To a drain terminal 2 of the ISFET is connected the positive potential of a voltage source Vd, and to its source terminal 3 is connected a constant current source 5. The ISFET operates through a source follower circuit thus formed. Any change in the conductivity of the channel under the ion sensitive membrane that results from a change in the boundary electric potential between the membrane 7 and the to-be-assayed solution 8 is drawn in terms of source potential Vs. Since the source potential Vs is in linear relation to a logarithm of ion concentration, it is possible to measure the concentration of ions contained in the solution being assayed by determining the source potential Vs.
In order to measure the quantity of chemical substances contained in the solution being assayed by using said ISFET or the conventional glass electrode as a potentiometric electrode for chemical-substance measurement, there must be a reference electrode having a constant potential in its interface with the being-assayed solution regardless of the composition of the solution. Therefore, the reference electrode is of such construction that the to-be-assayed solution may be brought into contact with an internal solution housed in a closed tube and having a constant composition, through a small space called liquid junction which is provided at front end of the tube. In the internal solution within the tube there is immersed an internal electrode such as Ag-AgCl electrode.
A reference electrode used together with ion sensors, such as ISFET, particularly for the purpose of living-body monitoring must be miniaturized enough to be insertible in a living body and must be steam-autoclavable. In the case of a combined-type sensor having an ion sensor and a reference electrode housed together in one tube, it is required that the reference electrode be further miniaturized. However, the smaller the reference electrode in size, the less is the quantity of the internal solution housed in the tube. In a prolonged measurement operation, therefore, it is very important to prevent the outflow of the internal solution. In order to reduce the flowout of the internal solution, there has been proposed a reference electrode having its liquid junction comprised of an acetyl cellulose membrane or ceramic which is permeable to water and ions and yet retards the diffusion thereof. Such reference electrode has the advantage that the outflow of the internal solution during a measurement operation can be reduced, but on the other hand it has the following difficulties during steam autoclavation (which is usually carried out in a hot, high-pressure steam atmosphere at about 120.degree. C. for approximately 20 minutes):
(1) that the internal solution flows out in the course of steam autoclavation; and
(2) that air dissolved in the internal solution adheres to the surface of the internal electrode in the form of bubbles, or interrupts the conduction between the internal electrode and the solution being assayed, which leads to an increased electric resistance between the solution and the internal electrode and causing instability of the reference electrode potential.
In order to overcome these difficulties, a usual practice is that an internal solution steam-autoclaved before use is placed in the tube in which the internal electrode is housed. While the difficulties discussed involved in above with respect to and steam autoclavation can be overcome, this practice is not desirable since it involves the possibilities that the effect of sterilization may be impaired unless extreme care is used when the sterilized internal solution is placed into the tube, care must be taken to prevent air from being mixed into the internal solution when the latter. It is also known to use sols of agar-agar, gelatine, polyvinyl alcohol, polyhydroxymethyl methacrylate, and the like, or hydrogels of hyperfine silica and the like, for the internal solution in order to prevent the outflow of the internal solution during measurement. Such reference electrode having such sol or hydrogel contained therein as the internal solution involves maintenance difficulties, though it is effective to some extent for the prevention of outflow of the internal solution in the course of measurement and also for the prevention of bubble generation in the internal solution during storage. Further, sols of organic polymers such as, for example, agar-agar, gelatine, polyvinyl alcohol, and polyhydroxy methacrylate, when heated, produce a potassium permanganate reducing substance which may be hazardous to living bodies. Polyhydroxyethyl methacrylate sol cannot be used as the internal solution because it is liable to separate into a polymer phase and a water phase upon heating. Hyperfine silica hydrogel is so high in viscosity that it is difficult, if not impossible, to put the gel into the tube.
In view of these facts, the present inventors proposed in Japanese Published Unexamined Patent Application No. 204363/1983 a steam autoclavable, miniaturized liquid-junction type reference electrode having its internal solution gelled by a high polymer.
Said reference electrode comprises: a one piece tube, an internal solution housed in this tube and gelled by 2.about.10% by weight of polyvinyl alcohol and 0.1.about.2% by weight of a crosslinking agent selected from the group consisting of titanium compounds, zirconium compounds, and vanadium compounds, an internal electrode immersed in the internal solution, and a liquid junction provided in the front end portion of the tube and with communicating between the inside of the tube and the outside thereof.
However, subsequent studies made with said reference electrode, in which aforesaid polymeric gel was used as a holder for the internal solution, revealed that the reference electrode still involved a number of problems yet to be solved, as enumerated below.
(1) During storage or steam autoclavation, bubbles in the polymeric gel aggregate into larger bubbles, whereby the electric connection between the internal electrode and the solution to be assayed is broken.
(2) Use of the polymeric gel as the holder for the internal solution causes a large liquid junction potential, with the result that the potential of the reference electrode varies according to the composition of the solution to be assayed.
(3) The manufacture of the reference electrode is complicated because the internal solution has to be gelled by the polymer and crosslinking agent in the reference electrode.